Mammalian erythropoiesis, as assayed by erythroid colony formation in vitro, is enhanced by cyclic adenosine nucleotides and agents which are capable of raising intracellular cyclic AMP (cAMP) levels. With canine marrow cells as target, this enhancement was shown to be specific for cAMP and its mono- and dibutyryl derivatives. Adenosine and its derivatives, such as AMP, ADP and ATP, and other cyclic nucleotides, such as cGMP, dibutyryl-cGMP, cCMP and cIMP and sodium butyrate were inactive. The phosphodiesterase inhibitor, RO-20-1724, and the adenyl cyclase stimulator, cholera enterotoxin, both markedly increased colony numbers. Studies with tritiated thymidine showed that about 50% of the cells responding to either erythropoietin (ESF) or dibutyryl cAMP (db-cAMP) were in DNA synthesis. However, by unit gravity sedimentation velocity analysis, the peak of ESF-responsive colony forming cells sedimented more rapidly (8-7 +/- 0-2 mm/hr) than the peak of db-cAMP-responsive cells (7-5 +/- 0 mm/hr). These results demonstrate that adenyl cyclase-linked mechanisms influence in vitro erythropoietic proliferation and suggest that other hormones and simple molecules might interact with surface receptors and thus modulate the action of ESF at the cellular level.